Viruses need to infect their hosts’ cells in order to replicate. But the hosts tend to not want these pathogens utilizing their resources, killing their cells, and putting their lives in danger. As such, the evolution of both host and pathogen have resulted in strategies that attempt to ‘outsmart’ the other; viruses can develop mechanisms to escape detection or replicate more rapidly, hosts can develop systems to detect pathogens in novel ways or enhance the function of their immune responses towards these invaders. This ‘evolutionary arms race’ is driven by mutations in genes which are associated with host-pathogen interactions. In humans, for instance, restriction factors, which are important in antiviral defenses, have been seen to evolve rapidly, primarily through the accumulation of missense mutations (swapping out of a single nucleotide for another and resulting in a change in the corresponding amino acid). Another common type of mutation which can drive evolutionary adaptations is referred to as indels (insert/deletions), but which often result in negative effects on protein function. Yet, despite a general consensus of indels being primarily detrimental, a positive impact of indels on anti-viral protein evolution was the main focus of the manuscript published in BioRxiv by Dr. Jeanette Tenthorey, formerly in the Malik Lab in the Division of Basic Sciences and now an Assistant Professor at UCSF.
The research team had previously published results showing that a single, specific, missense mutation in the human restriction factor TRIM5a was able to provide resistance to HIV infection as well as other retroviruses, except for one: simian lentivirus endemic in sabaeus monkeys (SIVsab). “This difference is what intrigued us and caused us to dig deeper on its evolutionary trajectory,” Dr. Tenthorey explained. They first wanted to assess whether more distinct missense mutations might be able to provide this resistance, and so the team generated a library of all the possible missense mutations in the region of the human TRIM5a which is most important for its specificity towards lentiviruses, the v1 loop. They expressed these sequences individually in cells which lacked their own TRIM5a, and then infected these cells with a SIVsab that also expressed GFP, so that they could detect cells which were resistant to infection as being GFP negative. However, what they observed was no single missense mutation was able to overcome this evolutionary hurdle of resisting SIVsab infection. They also compared the human TRIM5a v1 loop to that of the rhesus TRIM5 (which does resist SIVsab infection) and asked whether the nine differences between species in this region, individually or in combinations, could mimic the effect if the full rhesus v1 loop. They did find that a pattern of five specific mutations (four substitutions and one insertion) that were enriched in their restrictor-pool of variants, although each variant had additional, unique, mutations present. and when these positions were mutated in the original rhesus TRIM5a sequence, the team saw reduced resistance to SIVsab. However, they found that when they engineered human cells to express a variant expressing the five mutations, they were not sufficient for resistance, and only in the case of an additional sixth mutation did they observe strong resistance to SIVsab. The team concluded that the chances of six independent mutations at specific points in the genetic sequence would be extremely low.
However, Dr. Tenthorey explained that they uncovered a potential alternative route for the evolution of this protein towards better resistance to SIVsab. The research team saw an association of indels in the human TRIM5a with the accumulation of missense mutations, prompting them to consider “whether indels might have a similar impact on acquisition of functional novelty [as missense mutations],” explained Dr. Tenthorey. The team was initially not interested in indel mutations, as they are often considered as having negative impacts in protein function, and so they had to shift their perspective on how indels may be contributing positively in this context. They generated another library, this time of different indel mutations in the TRIM5a v1 loop which they screened for SAVsab restriction, as they had done previously. This time, they quickly found three indels which had strong restriction function, including a variant which only had one amino acid duplication. Dr. Tenthorey emphasized their surprise, “we were...shocked that a simple indel mutation could confer human TRIM5a with this antiviral function, when it was so hard for us to engineer it otherwise.” The team was excited by this finding and noted that there appeared to be three indel mutations that had occurred within just a short sequence of TRIM5a alone. They hypothesized that TRIM5a along with other restriction factors may utilize indels for rapid evolutionary changes that could confer protection against pathogens.
The team is encouraged to see how indels may affect other proteins in ways that have previously been overlooked or underappreciated. “As a start, we need to re-evaluate the role of indel mutations in a variety of antiviral proteins,” Dr. Tenthorey shared, “…Clearly, we should be looking for the accumulation of indels and considering these as potential avenues to new protein functions.” While indels are still considered to be primarily detrimental to protein function, they are still found concentrated within certain regions of genomes, suggesting that that they could potentially provide advantages to protein functions. The study here highlights that indels may even have significant advantages over missense mutations in certain genes where these more common evolutionary drivers cannot surmount fitness disadvantages. In addition to their overall excitement from their findings, Dr. Tenthorey also emphasized her immense appreciation for her research team, both here at the Hutch, as well as at her new home institution at UCSF, whom without, none of this hard work would have been possible.
This spotlight work was funded by the Howard Hughes Medical Institute, National Institutes of Health, American Foundation for AIDS Research, and the Mathilde Krim Fellowship in Biomedical Research.
Fred Hutch/University of Washington/Seattle Children’s Cancer Center Consortium members Drs. Michael Emerman and Harmit Malik contributed to this work.
Tenthorey JL, Del Banco S, Ramzan I, Klingenberg H, Liu C, Emerman M, Malik HS. 2024. Indels allow antiviral proteins to evolve functional novelty inaccessible by missense mutations. bioRxiv. [Preprint].